Oleaginous yeast are defined as those organisms that are naturally capable of oil synthesis and accumulation, wherein oil accumulation can be up to about 80% of the cellular dry weight. Genera typically identified as oleaginous yeast include, but are not limited to: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. More specifically, illustrative oil-synthesizing yeast include: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus, Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporon pullans, T. cutaneum, Rhodotorula glutinus, R. graminis and Yarrowia lipolytica (formerly classified as Candida lipolytica).
The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)). And, these organisms have been commercially used for a variety of purposes in the past. For example, various strains of Yarrowa lipolytica have historically been used for the manufacture and production of: isocitrate lyase; lipases; polyhydroxyalkanoates; citric acid; erythritol; 2-oxoglutaric acid; γ-decalactone; γ-dodecalactone; and pyruvic acid. Most recently, however, the natural abilities of oleaginous yeast have been enhanced by advances in genetic engineering, resulting in organisms capable of producing polyunsaturated fatty acids (“PUFAs”). Specifically, Picataggio et al. have demonstrated that Yarrowia lipolytica can be engineered for production of ω-3 and ω-6 fatty acids, by introducing and expressing genes encoding the ω-3/ω-6 biosynthetic pathway (co-pending U.S. patent application Ser. No. 10/840,579 and No. 60/624,812, herein incorporated by reference in their entirety).
Recombinant production of any heterologous protein is generally accomplished by constructing an expression cassette in which the DNA coding for the protein of interest is placed under the control of appropriate regulatory sequences (i.e., promoters) suitable for the host cell. The expression cassette is then introduced into the host cell (usually by plasmid-mediated transformation or targeted integration into the host genome) and production of the heterologous protein is achieved by culturing the transformed host cell under conditions necessary for the proper function of the promoter contained within the expression cassette. Thus, the development of new host cells (e.g., oleaginous yeast) for recombinant production of proteins generally requires the availability of promoters that are suitable for controlling the expression of a protein of interest in the host cell.
A variety of strong promoters have been isolated from Saccharomyces cerevisiae that are useful for heterologous gene expression in yeast. For example, a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was described by Bitter, G. A., and K. M. Egan (Gene 32(3):263-274 (1984)); and, a phosphoglycerate mutase (GPM1) promoter was investigated by Rodicio, R. et al. (Gene 125(2): 125-133 (1993)).
Several promoters have also been isolated from Yarrowia lipolytica that have been suitable for the recombinant expression of proteins. For example, U.S. Pat. No. 4,937,189 and EP220864 (Davidow et al.) disclose the sequence of the XPR2 gene (which encodes an inducible alkaline extracellular protease) and upstream promoter region for use in expression of heterologous proteins. However, this promoter is only active at a pH above 6.0 on media lacking preferred carbon and nitrogen sources; and, full induction requires high levels of peptone in the culture media. Subsequent analysis of the XPR2 promoter sequence by Blanchin-Roland, S. et al. (EP832258; Mol. Cell Biol. 14(1):327-338 (1994)) determined that hybrid promoters containing only parts of the XPR2 promoter sequence may be used to obtain high level expression in Yarrowia, without the limitations resulting from use of the complete promoter sequence.
U.S. Pat. No. 6,265,185 (Muller et al.) describe yeast promoters from Yarrowia lipolytica for the translation elongation factor EF1-α (TEF) protein and ribosomal protein S7 that are suitable for expression cloning in yeast and heterologous expression of proteins. These promoters were improved relative to the XPR2 promoter, when tested for yeast promoter activity on growth plates (Example 9, U.S. Pat. No. 6,265,185) and based on their activity in the pH range of 4-11.
Finally, work performed in conjunction with the Applicants herein has also resulted in the identification and isolation of the Yarrowia lipolytica fructose-bisphosphate aldolase promoters (e.g., FBA, FBAIN and FBAINm; see co-pending U.S. patent application Ser. No. 10/869,630, incorporated by reference herein) and the Yarrowia lipolytica glycerol-3-phosphate O-acyltransferase promoter (GPAT; see co-pending U.S. Patent Application No. 60/610,060, incorporated by reference herein).
Despite the utility of these known promoters, however, there is a need for new improved yeast regulatory sequences for metabolic engineering of yeast (oleaginous and non-oleaginous) and for controlling the expression of heterologous genes in yeast. Furthermore, possession of a suite of promoters that are regulatable under a variety of natural growth and induction conditions in yeast will play an important role in industrial settings, wherein it is desirable to express heterologous polypeptides in commercial quantities in said hosts for economical production of those polypeptides. Thus, it is an object of the present invention to provide such regulatory sequences that will be useful for gene expression in a variety of yeast cultures, and preferably in Yarrowia sp. cultures and other oleaginous yeast.
Applicants have solved the stated problem by identifying genes encoding a glyceraldehyde-3-phosphate dehydrogenase (GPD) and a phosphoglycerate mutase (GPM) from Yarrowia lipolytica and the regulatory sequences responsible for driving expression of these native genes. Specifically, the GPD promoter, GPDIN promoter, GPD intron, GPD enhancer and GPM promoter are each useful for expression of heterologous genes in Yarrowia and have improved activity with respect to the TEF promoter.